Multiple sclerosis (MS) is a chronic neurological disease of unknown etiology, but a genetic basis for the disease is undisputed. We have reported that CD24 is required for the pathogenicity of autoreactive T cells in experimental autoimmune encephalomyelitis, the mouse model of MS. Here we investigate the contribution of CD24 to MS by studying single-nucleotide polymorphism in the ORF among 242 MS patients and 207 population controls. This single-nucleotide polymorphism results in replacement of alanine (CD24 a ) with valine (CD24 v ) in the mature protein. We found that the CD24 v/v renders a >2-fold increase in the relative risk of MS in the general population (P ‫؍‬ 0.023). Among familial MS, the CD24 v allele is preferentially transmitted into affected individuals (P ‫؍‬ 0.017). Furthermore, 50% of CD24 v/v patients with expanded disability status scale 6.0 reached the milestone in 5 years, whereas the CD24 a/v (P ‫؍‬ 0.00037) and CD24 a/a (P ‫؍‬ 0.0016) patients did so in 16 and 13 years, respectively. Moreover, our data suggest that the CD24 v/v patients expressed higher levels of CD24 on peripheral blood T cells than did the CD24 a/a patients. Transfection with CD24 a and CD24 v cDNA demonstrated that the CD24 v allele can be expressed at higher efficiency than the CD24 a alleles. Thus, CD24 polymorphism is a genetic modifier for susceptibility and progression of MS in the central Ohio cohort that we studied, perhaps by affecting the efficiency of CD24 expression on the cell surface.single-nucleotide polymorphism ͉ disease susceptibility ͉ autoimmunity ͉ costimulatory molecules ͉ T lymphocytes M ultiple sclerosis (MS) is a chronic disorder in the CNS that affects Ϸ0.1% of Caucasians of northern European origin (1). The incidence of MS is increased among family members of affected individuals. The concordance rate of the identical twins can be as high as 30% (1-3). The HLA loci is perhaps the most important genetic element for MS susceptibility, because the HLA-DR2 allele has been identified as the most important susceptibility gene among Caucasians (4-10). Several additional loci have also been proposed (8-12).One of the whole-genome scans suggested a linkage disequilibrium in distal 6q (8) whose identity has not been revealed. An interesting candidate in the region is CD24 (13), which we showed to be essential for the induction of experimental autoimmune encephalomyelitis (EAE) in mice (13). CD24 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein with expression in a variety of cell types that can participate in the pathogenesis of MS, including activated T cells (14, 15), B cells (16), macrophages (17), dendritic cells (18), and local antigen-presenting cells in the CNS, such as vascular endothelial cells, astrocytes, and microglia (our unpublished observation). It is well established that in the mouse CD24 mediates a CD28-independent costimulatory pathway that promotes activation of CD4 and CD8 T cells (16)(17)(18)(19)(20)(21). In addition, CD24 has been shown to modulate the very l...

In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen (CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or nonhematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE.

It is generally believed that susceptibility to both organ-specific and systemic autoimmune diseases is under polygenic control. Although multiple genes have been implicated in each type of autoimmune disease, few are known to have a significant impact on both. Here, we investigated the significance of polymorphisms in the human gene CD24 and the susceptibility to multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We used cases/control studies to determine the association between CD24 polymorphism and the risk of MS and SLE. In addition, we also considered transmission disequilibrium tests using family data from two cohorts consisting of a total of 150 pedigrees of MS families and 187 pedigrees of SLE families. Our analyses revealed that a dinucleotide deletion at position 1527∼1528 (P1527del) from the CD24 mRNA translation start site is associated with a significantly reduced risk (odds ratio = 0.54 with 95% confidence interval = 0.34–0.82) and delayed progression (p = 0.0188) of MS. Among the SLE cohort, we found a similar reduction of risk with the same polymorphism (odds ratio = 0.38, confidence interval = 0.22–0.62). More importantly, using 150 pedigrees of MS families from two independent cohorts and the TRANSMIT software, we found that the P1527del allele was preferentially transmitted to unaffected individuals (p = 0.002). Likewise, an analysis of 187 SLE families revealed the dinucleotide-deleted allele was preferentially transmitted to unaffected individuals (p = 0.002). The mRNA levels for the dinucleotide-deletion allele were 2.5-fold less than that of the wild-type allele. The dinucleotide deletion significantly reduced the stability of CD24 mRNA. Our results demonstrate that a destabilizing dinucleotide deletion in the 3′ UTR of CD24 mRNA conveys significant protection against both MS and SLE.

It is well established that B7-CD28/CTLA4 interactions play an important role in the induction of T helper cells for T-dependent antibody responses. However, targeted mutation of CD28 does not significantly affect production of IgG and activation of CD4 T helper cells in response to infections by some viruses and nematode parasites. To test whether the CD28-independent induction of Ig class switches requires costimulation by the heat-stable antigen (HSA), we compared T helper cell induction and antibody response in mice deficient for either HSA, CD28, or both genes. We found that after immunization with KLH-DNP, mice deficient for both CD28 and HSA lack DNP-specific IgA and all subtypes of IgG. This deficiency corresponds to a reduced number of effector helper T cells that rapidly produce IL-2, IL-4, and IFN-γ after in vitro stimulation with carrier antigen KLH. In contrast, priming of T helper cells and Ig class switch are normal in mice deficient with either HSA or CD28 alone. IgM responses are not affected by any of these targeted mutations. These results demonstrate that CD28-independent induction of T helper cells and Ig class-switches requires costimulation by the HSA.

The heat-stable antigen (HSA) is an important co-stimulatory molecule on antigen-presenting cells (APC). However, the receptor on T cells that receives the co-stimulatory signal from HSA has not been identified. Because the HSA is transiently expressed on T cells after the T cell receptor/CD3 complex is engaged, and because it can bind to itself in a homotypic fashion, it has been proposed that homotypic interaction of HSA is responsible for its co-stimulatory activity. Here we test this hypothesis using mice that have a targeted mutation of the HSA gene, as well as novel transgenic mice that constitutively express HSA on T cells. We show that HSA-deficient T cells remain responsive to co-stimulation by HSA. Furthermore, constitutive expression of HSA does not enhance T cell response to co-stimulatory by HSA. Taken together, our results demonstrate that homotypic interaction of HSA is not responsible for co-stimulation mediated by HSA expressed on APC.

The heat-stable antigen (HSA) is a costimulatory molecule for T-cell activation. Its expression is strictly regulated during lymphocyte development and differentiation. Recent studies using HSA-transgenic mice have demonstrated that this regulated expression is critical for normal development of T and B lymphocytes. However, the mechanisms that control the expression of HSA are largely unknown. HSA mRNA is comprised of a 0.23-kb open reading frame and a 1.5-kb 3 untranslated region (3UTR). The function of the long 3UTR has not been addressed. Here we investigate the role of the 3UTR of HSA mRNA. We show that a 160-bp element, located in the region of nucleotides 1465 to 1625 in the 3UTR of HSA mRNA, promotes RNA degradation and that this effect is neutralized by a 43-bp fragment approximately 1 kb upstream of the negative cis element. Both positive and negative cis elements in the HSA mRNA are distinct from other sequences that are known to modulate mRNA stability. These results provide direct evidence that the interplay between two novel cis elements in the 3UTR of HSA mRNA determines cell surface HSA expression by modulating its RNA stability.

Transporter associated with antigen processing (TAP), a member of the ATP-binding cassette transporter superfamily, is composed of two integral membrane proteins,TAP-1and TAP-2. Each subunit has a C-terminal nucleotide-binding domain that binds and hydrolyzes ATP to energize peptide translocation across the endoplasmic reticulum membrane. A motif comprising the sequence LSGGQ (called the signature motif) and the amino acid that is immediately C-terminal to this motif are highly conserved in the nucleotide-binding domains of ATP-binding cassette transporters. To search for natural variants of TAP-1with alterations in or near the signature motif, we sequenced the TAP-1 exon 10 amplified from 103 human colon cancer samples. We found a rareTAP-1 allele with an R>Q alteration at a residue immediately C-terminal to the signature motif (R648) that occurred 17.5 times more frequently in colon cancers with down-regulated surface class I MHC than those with normal MHC levels (P = 0.01). Functional analysis revealed that the Q648 variant had significantly reduced peptide translocation activity compared with TAP-1(R648). In addition, we found that mutations S644R, G645R, G646S, and G646D interfered withTAP-1activity. TAP-1G646D, which showed the most severe defect, resided normally in the endoplasmic reticulum and associated with the peptide loading complex, but failed to transport peptide across the endoplasmic reticulum membrane. Thus, a TAP-1 polymorphism adjacent to the signature motif may be a contributing factor for MHC class I down-regulation in colon cancer. Given the widespread defects in DNA mismatch repair in colon cancer, mutations at or near the signature domain can potentially modulate antigen processing.

Placental growth factor play (PLGF) is a homolog of vascular endothelial growth factor (VEGF) and binds to VEGF receptor-1. Previous studies have indicated its synergistic role with VEGF in angiogenesis, while a recent study suggested its independent role in promoting growth and metastasis of VEGF-inhibitor-resistant tumors. In order to further investigate the role of PLGF in tumor angiogenesis in relationship to VEGF, we have generated a novel PLGF-specific monoclonal antibody (mAb). Hybridomas secreting PLGF-specific mAbs were obtained by fusing splenocytes from mice immunized with yeast-derived recombinant human PLGF protein with Sp/20 myeloma cells; the mAb clone that showed highest binding affinity, designed as P16, was selected for further study. This P16 mAb recognizes human PLGF, in both native and denatured forms, but not VEGF-A, VEGF-B or VEGF-D. P16 mAb is of IgG1-kappa type. Sequence analysis of cDNA encoding the heavy and light chains of P16 mAb showed novel variable regions. Using P16 mAb in immunohistochemical staining assay, increased levels of PLGF protein were found in a number of human tumors from lung, breast and colon. PLGF proteins were also detected in a wide variety of normal human tissues including lung, stomach, colon and kidney, contrary to previous reports. Importantly, P16 mAb blocked the binding of PLGF to VEGFR-1 in a dose-response manner, and inhibited the PLGF-mediated endothelial cell line growth in vitro. Treatment of human tumor xenografts (A673 rhabdomyosarcoma) in nude mice with P16 mAb resulted in marked inhibition of tumor growth, which is similar in degree to the tumor inhibition by VEGF-specific antibody Bevacizumab. A humanized form of P16 mAb was constructed and showed similar inhibition of tumor growth in xenograft nude mice model. In conclusion, P16 mAb is a novel mAb specifically recognizing human PLGF but not VEGF and blocks the binding of PLGF to its receptor. It inhibits angiogenesis and tumor growth, suggesting an important and independent role of PLGF in tumor growth. Further studies on its mechanisms of action and its clinical applications are under way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2453.